Inhibition of lipoprotein oxidation

ABSTRACT

Hydroxylated derivatives of cholesterol lowering agents inhibit the oxidation of lipoproteins, and are thus useful for preventing the progression of atherogenesis and resultant vascular diseases, including heart attacks.

This application is a 371 of PCT/U.S. 98/23483 filed Nov. 4, 1998 whichclaim benefit of Provisional No. 60/066,888 filed Nov. 25, 1997.

FIELD OF THE INVENTION

This invention relates to a method for inhibiting the oxidation oflipoproteins, thereby slowing or stopping atherogenesis. The methodentails the use of hydroxylated derivatives of known cholesterollowering agents.

BACKGROUND OF THE INVENTION

Atherosclerotic cardiovascular diseases and related conditions anddisease events associated with hyperlipidemia are major causes ofdisability and death. It is now well recognized that lowering certainforms of cholesterol, both in healthy mammals as well as in individualsalready experiencing states of hyperlipidemia, can dramatically reduceheart attacks, vascular disease, and other diseases associated withatherosclerotic conditions.

Hyperlipidemia is a condition which is characterized by an abnormalincrease in serum lipids, such as cholesterol, triglycerides, andphospholipids. These lipids do not circulate freely in solution inplasma, but are bound to proteins and transported as macromolecularcomplexes called lipoproteins. There are five classifications oflipoproteins based on their degree of density: chylomicrons, very lowdensity lipoproteins (VLDL), low density lipoproteins (LDL),intermediate density lipoproteins (IDL), and high density lipoproteins(HDL).

One form of hyperlipidemia is hypercholesterolemia, characterized by theexistence of elevated LDL cholesterol levels. The initial treatment forhypercholesterolemia is often to modify the diet to one low in fat andcholesterol, coupled with appropriate physical exercise, followed bydrug therapy when LDL-lowering goals are not met by diet and exercisealone. LDL is commonly known as the “bad” cholesterol, while HDL is the“good” cholesterol. Although it is desirable to lower elevated levels ofLDL cholesterol, it is also desirable to increase levels of HDLcholesterol. Generally, it has been found that increased levels of HDLare associated with lower risk for coronary heart disease (CHD).

While LDL cholesterol is recognized as bad, and mostcholesterol-lowering agents operate by lowering the plasma concentrationof the LDL form, there is another key process in the early stages ofatherogenesis, that being oxidation of LDL. Oxidation of VLDL and HDLalso occurs, which also contributes to atherogenesis. Oxidation leads toincreased intracellular calcium, lowered energy production, activationof cytokines, membrane damage, all resulting in apoptosis, necrosis, andultimately cell death.

Oxidation typically begins when a reactive radical abstracts a hydrogenatom from a polyunsaturated fatty acid on the LDL particle. Lipidperoxyl and alkoxyl radicals are formed, which in turn can initiateoxidation in neighboring fatty acids, resulting in propogation of lipidperoxidation. These oxidized forms of lipoproteins are absorbed bymacrophages more rapidly than the native lipoproteins, and this resultsin macrophage cholesterol accumulation, and subsequent foam cellformation and inhibition of the motility of tissue macrophages andendothelial cells. This cascade of events results in vasculardysfunction and formation and activation of atherosclerotic lesions.

We have now discovered that certain hydroxy-substituted derivatives ofcommonly employed cholesterol lowering agents are effective antioxidantsfor lipoproteins. Additionally, these compounds are useful for freeradical scavenging and metal ion chelation, which also are mechanisms bywhich lipoproteins are oxidized.

SUMMARY OF THE INVENTION

This invention provides a method for inhibiting oxidation oflipoproteins in a mammal comprising administering an antioxidanteffective amount of a hydroxylated cholesterol lowering agent. Theinvention also provides a method for scavenging free radicals in amammal comprising administering a free radical scavenging amount of ahydroxylated cholesterol-lowering agent. The invention also provides amethod for inhibiting metal ion chelation by lipoproteins comprisingadministering an effective amount of a hydroxylated cholesterol loweringagent.

In a preferred embodiment, the methods are practiced utilizing ahydroxylated form of a statin, especially atorvastatin, which compoundsare described in U.S. Pat. No. 5,385,929, which is incorporated hereinby reference.

In another preferred embodiment, the methods are practiced utilizing ahydroxylated gemfibrozil, e.g., a compound of the formula

in another embodiment, a hydroxylated fluvastatin is employed, e.g.,compounds of the formula

In another embodiment, a hydroxylated cerivastatin is employed,especially a compound of the formula

In another preferred embodiment, hydroxylated derivatives of lovastatinare employed, e.g., compounds of the formulas

BRIEF DESCRIPTION OF THE FIGS.

FIG. 1. Structural formulas of atorvastatin and its hydroxylatedmetabolites.

FIG. 2. Structural formulas of gemfibrozil and its hydroxylatedmetabolite.

FIG. 3. The effect of atorvastatin and its hydroxylated metabolites onLDL oxidation in the copper ion oxidative system (A), the AAPH oxidativesystem (B), and the J-774 A.1 macrophage oxidative system (C). LDL (100μg of protein/mL) was incubated in all three oxidative systems withincreasing concentration of the drug or its metabolites for 4 hours at37° C. in systems A and B, and for 20 hours with the cells (C). At theend of the incubation, LDL oxidation was measured by the TBARS assay.Macrophage-mediated oxidation of LDL was calculated by subtraction ofthe values obtained in the absence of cells from those obtained in thepresence of cells. Results are given as the mean±standard deviation (SD)(n=3).

FIG. 4. The effect of atorvastatin and its hydroxylated metabolites onVLDL oxidation in the copper ion oxidative system (A), and in AAPHoxidation system (B). VLDL (100 μg of protein/mL) was incubated with 10μM of atorvastatin or its metabolites for 4 hours at 37° C. At the endof the incubation, VLDL oxidation was measured by the TBARS assay.Results are given as mean±SD (n=3).

FIG. 5. Free radical scavenging activity (A), and copper ion chelatingcapability of atorvastatin and its hydroxylated metabolites (B). A.Atorvastatin or its hydroxylated metabolites (20 μM) were incubated with1 mM DPPH and kinetic determination of the absorbance at 517 nm wasperformed. A representative experiment out of 3 different studies withsimilar pattern is shown. Vitamin E (20 μM) was used as a positivecontrol for free radicals scavenger. B. LDL (100 μg of protein/mL) wasincubated with atorvastatin or its metabolites (10 μM) and withincreasing concentrations of CuSO₄ for 4 hours at 37° C., prior, toanalysis of lipoprotein oxidation by the TBARS assay. Results are givenas mean±SD (n=3).

FIG. 6. Effect of gemfibrozil and gemfibrozil metabolite concentrationon LDL oxidation in the copper ion oxidative system (A), the AAPHoxidative system (B), and J-774 A.1 macrophage oxidative system (C). LDL(100 μg of protein/mL) was incubated in all three oxidative systems withincreasing concentrations of the drug or its metabolite, for 4 hours at37° C. in systems A and B, and for 20 hours with the cells (C). At theend of the incubation, LDL oxidation was measured by the TBARS assay.Macrophage-mediated oxidation of LDL was calculated by subtracting thevalues obtained in the absence of cells from those obtained in thepresence of cells. Results are given as the mean±SD (n=3).

FIG. 7. The effect of gemfibrozil and its metabolite on VLDL oxidationin copper ion oxidative system (A) and in the AAPH oxidation system (B).VLDL (100 μg of protein/mL) was incubated with 4 μM of gemfibrozil orits metabolite for 4 hours at 37° C. At the end of the incubation, VLDLoxidation was measured by the TBARS assay. Results are given as mean±SD(n=3).

FIG. 8. Lipoprotein electrophoresis of VLDL following copper ions (10 μMCuSO₄)-induced lipoprotein oxidation in the absence or presence ofatorvastatin, gemfibrozil, or their metabolites.

FIG. 9. Free radical scavenging activity (A), and copper ion chelatingcapability of gemfibrozil and its metabolite (B). A. Gemfibrozil or itsmetabolite (20 μM) were incubated with 1 mM DPPH and kineticdetermination of the absorbance at 517 mm was performed. Arepresentative experiment out of 3 different studies with similarpattern is shown. Vitamin E (Vit E) at similar concentration was used asa control free radical scavenger. B. LDL (100 μg of protein/mL) wasincubated with gemfibrozil or its metabolite (3 μM) and with increasingconcentrations of CuSO₄ for 4 hours at 37° C. prior to analysis oflipoprotein oxidation by the TBARS assay. Results are given as mean±SD(n=3).

FIG. 10. The combined effect of metabolite I of gemfibrozil and theortho-hydroxy metabolite of atorvastatin on LDL oxidation. LDL (100 μgof protein/mL) was incubated with 10 μM CuSO₄ for 4 hours at 37° C.alone (Control) or in the presence of gemfibrozil metabolite I (3 μM),or the atorvastatin ortho-hydroxy metabolite (4 μM) alone, or incombination. Lipoprotein oxidation was then measured by the TBARS assay.*p<0.01 (vs. Control) and p<0.01 (vs. Metabolite I), #P<0.01 (vs.Ortho-Hydroxy). Results are given as the mean±SD (n=3).

FIG. 11. The dose-dependent antioxidant effects of atorvastatinpara-hydroxy metabolite in membrane preparations enriched withpolyunsaturated fatty acids.

FIG. 12. The comparative antioxidant potency of atorvastatinpara-hydroxy metabolite, Vitamin E, and probucol.

FIG. 13. The antioxidant potency of atorvastatin para-hydroxy metaboliteand Vitamin e under atherosclerotic-like conditions of elevated membranecholesterol.

DETAILED DESCRIPTION OF THE INVENTION

The term “hydroxylated cholesterol lowering agent” means any chemicalcompound that is effective at lowering LDL cholesterol in a mammal thathas at least one hydroxy group substituted on the parent structure, andhas antioxidant activity. Examples include hydroxylated statins. Thestatins are a known class of HMG-CoA reductase inhibitors, such asatorvastatin, fluvastatin, and cerivastatin. Hydroxylated statins arethe parent statin compound having at least one hydroxy substituentgroup, examples being ortho-hydroxy atorvastatin and para-hydroxyatorvastatin as shown in FIG. 1. Other hydroxylated cholesterol loweringagents are hydroxy substituted fibrates, such as hydroxylatedgemfibrozil as shown in FIG. 2 (metabolite I). The hydroxylated compoundto be used in the method of this invention is preferably a compoundhaving a hydroxy group attached to a phenyl ring.

Increased atherosclerosis risk in hyperlipidemic patients results fromenhanced oxidizability of their plasma lipoproteins. Whilehypocholesterolemic drug therapy, including the3-hydroxy-3-methyl-glutaryl Coenzyme A (HMG-CoA) reductase inhibitorssuch as atorvastatin, and the hypotriglyceridemic drug bezafibrate,reduces the enhanced susceptibility to oxidation of low densitylipoprotein (LDL) isolated from hyperlipidernic patients, thisantioxidative effect could not be obtained in vitro with these drugs.The following experiments establish the effect of atorvastatin andgemfibrozil, as well as specific hydroxylated metabolites, on thesusceptibility of LDL, VLDL, and HDL to oxidation (e.g., lipidperoxidation). Lipid peroxidation, induced by either copper ions (10 μMCuSO₄), by the free radical generator system 2′2′-azobis 2-amidinopropane hydrochloride (5 mM AAPH), or by the J-774A.1 macrophage-likecell line, was not inhibited by the parent forms of atorvastatin orgemfibrozil, but was substantially inhibited (by 57%-97%), in aconcentration-dependent manner, by pharmacological concentrations of theortho-hydroxy and the para-hydroxy metabolites of atorvastatin, as wellas by para-hydroxy metabolite (metabolite I) of gemfibrozil. On usingthe atorvastatin ortho-hydroxy metabolite and gemfibrozil metabolite Iin combination, an additive inhibitory effect on LDL oxidizability wasfound. Similar inhibitory effects (37%-96%) of the above metaboliteswere obtained for the susceptibility of VLDL and HDL to oxidation in theoxidation systems outlined above. The inhibitory effects of thesemetabolites on LDL, VLDL, and HDL oxidation could be related to theirfree radical scavenging activity, as well as (mainly for the gemfibrozilmetabolite I) to their metal ion chelation capacities. In addition,inhibition of HDL oxidation was associated with preservation ofHDL-associated paraoxonase activity. The data establish thatatorvastatin hydroxy metabolites, and gemfibrozil metabolite I, possesspotent antioxidative potential, and as a result protect LDL, VLDL, andHDL from oxidation. The hydroxylated cholesterol lowering agents thusare useful to reduce the atherogenic potential of lipoproteins throughtheir antioxidant properties.

LDL oxidation is a key process in early atherogenesis and thus,inhibition of LDL oxidation is antiatherogenic. VLDL and HDL oxidationalso occurs during oxidative stress and also contributes toatherogenesis. Antioxidants are derived environmentally as well asgenetically. For example, dietary antioxidants, such as vitamin E,carotenoids, or polyphenolic flavonoids, associated with lipoproteins,protects them from oxidation. In addition, genetic factors, such asHDL-associated paraoxonase, also protects this lipoprotein from thedamage of oxidative stress. The enhanced susceptibility of LDL tooxidation derived from hypercholesterolemic patients is significantlyreduced by hypocholesterolemic therapy. Thus, hypolipidemic therapy maybe considered beneficial not only because of its effects on plasma VLDL,LDL, and HDL levels, but also since it can reduce the formation ofatherogenic oxidized lipoproteins.

The ex vivo inhibition of LDL oxidation has been shown following theadministration of the HMG-CoA reductase inhibitors lovastatin,simvastatin, pravastatin, or fluvastatin to hypercholesterolemicpatients. The inhibitory effect of these drugs on LDL oxidizability wassuggested to result from enhanced removal of plasma “aged LDL”, which ismore prone to oxidation than newly synthesized LDL. This effect would besecondary to the statin-induced stimulation of LDL receptor activity inliver cells and to inhibition of hepatic VLDL and LDL production.Metabolites of the parent statins, which are produced in the liverduring drug therapy, may also be involved mechanistically. The hepaticP450 drug metabolizing system activity participates in altering theparent statin structure, usually by hydroxylation. Indeed, all the abovestatins, with the exception of fluvastatin, did not demonstrate directantioxidant effects on in vitro LDL oxidation when tested atconcentrations comparable to the blood drug levels observed in treatedhypercholesterolemic subjects. Atorvastatin, a new inhibitor ofHMG-CoA-reductase, is the most effective statin for reducing both plasmatotal and LDL cholesterol levels. This compound also possessessignificant hypotriglyceridemic properties towards all lipoproteinfractions. Atorvastatin therapy increases LDL receptor activity andinhibits direct production of apolipoprotein B-100 containinglipoproteins. Both parent drug and its metabolites have relatively longcirculation half lives of 14 to 36 hours. Fibrate drugs may also affectthe susceptibility of lipoproteins to oxidation; for example,bezafibrate possesses such a capability. The fibric acid derivatives arelipid regulating drugs that promote the catabolism of triglyceride-richlipoproteins, secondary to the activation of lipoprotein lipase, and tothe reduction of apoC-III synthesis. Another fibrate, gemfibrozil hasbeen shown to not only reduce plasma triglycerides, but also to increaseplasma HDL concentration in humans and to reduce plasma lipoprotein (a)levels in male cynomolgus monkeys. In humans, gemfibrozil is metabolizedto gemfibrozil acyl glucuronides, and these metabolites are found in theplasma and urine of volunteers following treatment. The level of thepara-hydroxy metabolite of gemfibrozil (metabolite I) found in theplasma of gemfibrozil-treated rodents is much higher than that oftreated humans and likely reflects differences in dose and metabolism.We have now shown the effects of atorvastatin and gemfibrozil, as wellas specific hydroxylated metabolites (alone and in combination) on LDL,VLDL, and HDL susceptibility to oxidation. The results clearlydemonstrate inhibitory effects of the drug metabolites (but not of theparent drugs) on plasma lipoprotein oxidation individually, and anadditive effect, when combined. The data establish that the hydroxylatedderivatives are useful to prevent lipoprotein oxidation and therebyreduce their atherogenic potential.

The following detailed examples demonstrate the antioxidant activity ofvarious hydroxylated cholesterol lowering agents.

EXAMPLE 1

Materials—Atorvastatin and its ortho-hydroxy and para-hydroxymetabolites (FIG. 1), as well as gemfibrozil and its metabolite I (FIG.2) were synthesized by prior art methods. 2,2-Azobis 2-amidinopropanehydrochloride (AAPH) was purchased from Wako Chemical Industries, Ltd.(Osaka, Japan). 1,1-Diphenyl-2 picryl-hydrazyl (DPPH) was purchased fromSigma (St. Louis, Mo.).

Lipoproteins—Serum VLDL, LDL, and HDL were isolated from fastednormolipidemic volunteers. Lipoproteins were prepared by discontinuousdensity gradient ultracentrifugation. The lipoproteins were washed attheir appropriate densities (1.006 g/mL, 1.063 g/mL, and 1.210 g/mL,respectively), and dialyzed against 150 mM NaCI, (pH 7.4) at 4° C. Thelipoproteins were then sterilized by filtration (0.45 μM), kept undernitrogen in the dark at 4° C., and used within 2 weeks. Prior to theoxidation studies, the lipoproteins were dialyzed against PBS, EDTA-freesolution, pH 7.4 under nitrogen at 4° C. The lipoproteins were found tobe free of lipopolysaccharide (LPS) contamination when analyzed by theLimulus Amebocyte Lysate assay (Associated of Cape Cod, Inc; Woods Hole,Mass., USA). The lipoprotein protein content was determined by standardmethods.

Lipoprotein oxidation—Lipoproteins (100 μg of protein/mL) were incubatedwith 10 μM CuSO₄ or with 5 mM of AAPH for 4 hours at 37° C. AAPH is awater-soluble azo compound that thermally decomposes and generates watersoluble peroxyl radicals at a constant rate. Oxidation was terminated bythe addition of 10 μM of butylated hydroxytoluene (BHT) andrefrigeration at 4° C. The extent of lipoprotein oxidation was measuredby the thiobarbituric acid reactive substances (TBARS) assay, usingmalodialdehyde (MDA) for the standard curve. In addition, lipoproteinoxidation was also determined by the lipid peroxidation test thatanalyze lipid peroxides by their capacity to convert iodide to iodinewhich can be measured photometrically at 365 nm. The kinetics of LDLoxidation was continuously monitored by measuring the formation ofconjugated dienes as the increase in the absorbance at 234 nm.

LDL oxidation by macrophages—J-774 A.1 murine macrophages-like cell linewas purchased from the American Type Culture Collection (Rockville,Md.). The macrophages were grown in Dulbecco's Modified Eagles Medium(DMEM) supplemented with 5% heat inactivated fetal calf serum (FCS). Forthe lipoprotein oxidation studies, cells (1×10⁶/35 mm dish) wereincubated with LDL (100 μg of protein/mL) in RPMI medium (without phenolred) in the presence of 2 μM CuSO₄ for 20 hours at 37° C. in theincubator. Control LDL was also incubated in a cell-free system underthe same conditions. At the end of the incubation period, the extent ofLDL oxidation was measured in the medium (after centrifugation at 1000×gfor 10 minutes) by the TBARS assay. Cell-mediated oxidation of LDL wascalculated by subtracting the values obtained in the cell-free systemfrom those obtained with the cells.

Lipoprotein electrophoresis—Lipoproteins (100 μg protein/mL) wereincubated without or with the drugs followed by oxidation in thepresence of 10 μM CuSO₄. Then, electrophoresis of the lipoproteins wasperformed on 1% agarose using a Hydragel-Lipo kit (Sebia, France).

Free radical scavenging capacity—The free radical scavenging capacitiesof the drugs were analyzed by the 1,1-diphenyl-2-picryl-hydrazyl (DPPH)assay. Each drug (20 μM) was mixed with 3 mL of 0.1 nmol DPPH/1 (inethanol). The time course of the change in the optical density at 517 nmwas then kinetically monitored.

Paraoxonase activity measurements—The rate of hydrolysis of paraoxon wasassessed by measuring the formation of p-nitrophenol at 412 nm at 25° C.The basal assay mixture included 1.0 mM paraoxon and 1.0 mM CaCI₂ in 50mM glycine/NaOH pH 10.5. One unit of paraoxonase activity produces 1nmol of p-nitrophenol per minute.

Statistical analyses—The Student t-test was used in comparing two means,whereas analysis of variance (ANOVA) was used when more than two groupswere compared. Data are presented as mean±standard deviation (SD).

RESULTS

The effect of atorvastatin and its hydroxy metabolites, as well as thatof gemfibrozil and its metabolite, on the susceptibility of lipoproteinsto oxidation was studied in several oxidation systems including thosecontaining metal ions (10 μM CuSO₄), those have the capacity to generatefree radicals (5 mM AAPH), and those that mimic biological oxidation(J-774A.1 macrophage-like cell line).

Atorvastatin and lipoprotein oxidation—LDL oxidation was inhibited bythe ortho-hydroxy and para-hydroxy atorvastatin metabolites, but not byatorvastatin in all oxidative systems studied. These inhibitory effectswere concentration-dependent (FIG. 3). At 10 μM, both the ortho-hydroxyand the para-hydroxy metabolites inhibited LDL oxidation measured by theTBARS assay in the CuSO₄ system by 73% and 60%, respectively (FIG. 3A);in the AAPH system, by 44% and 34%, respectively (FIG. 3B); and in themacrophage system by 50% and 46%, respectively (FIG. 3C). At allconcentrations studied and in all oxidation systems, the ortho-hydroxymetabolite was a better LDL oxidation inhibitor than the para-hydroxymetabolite (FIG. 3). A more potent inhibitory effect of bothatorvastatin metabolites was obtained in the metal ion oxidation system(FIG. 3A), in comparison to that induced by the free radical generatingsystem (FIG. 3B). Similar results were obtained in the other oxidativesystems when LDL oxidation was determined by analyses oflipoprotein-associated peroxides. The ortho-hydroxy and para-hydroxymetabolites of atorvastatin reduced LDL-associated peroxides contentfrom 710±51 in control LDL, to 192±15 and 284±13 nmol/mg LDL protein inthe CuSO₄ system, respectively, and from 990±89 in control LDL, to554±32 and 624±38 nmol/mg LDL protein in the AAPH system, respectively.Furthermore, kinetic analysis of conjugated dienes formations at 234 nmduring copper ion (10 μM CuSO₄)-induced LDL oxidation, revealed that thelag time required for the initiation of LDL oxidation was 50±7 minutes(n=3) for either control or atorvastatin-treated LDL, whereas LDLconjugated dienes formation initiated only after 180±25 minutes (n=3)for both of the atorvastatin metabolites.

The effect of atorvastatin and its metabolites on VLDL oxidation isreported in FIG. 4. In the copper ion oxidative system, theortho-hydroxy and para-hydroxy metabolites (10 μM) inhibited lipoproteinoxidation by 79% and 37%, respectively (FIG. 4A), whereas atorvastatinitself had no effect. In the AAPH oxidative system, the inhibitoryeffects of these metabolites were only 43% and 16%, respectively FIG.4B), and again atorvastatin itself had no effect. Similar results werefound when VLDL oxidation was analyzed by peroxide formation. Theortho-hydroxy and the para-hydroxy metabolites of atorvastatin reducedVLDL-associated peroxide content from 1818±333 in control VLDL, to242±22 and 1088±310 nmol/mg VLDL protein in the CuSO₄ system,respectively, and from 2169±329 in control VLDL, to 1228±210 and1819±228 nmol VLDL protein in the AAPH system, respectively. Similarly,HDL oxidation in the presence of CuSO₄ under similar incubationconditions revealed that the ortho-hydroxy metabolite completelyinhibited HDL oxidation, whereas the para-hydroxy metabolite inhibitedthe lipoprotein oxidation by about 50% (Table 1). The inhibitory effectsof these metabolites on HDL oxidation were associated with theprotection of paraoxonase by 54% and 27%, respectively. Elevatedactivities of the HDL-associated paraoxonase were noted, in comparisonto paraoxonase activity in HDL that was oxidized in the absence of addedparent drug (Table 1).

TABLE 1 The Effect of Atorvastatin and Its Metabolites on HDL Oxidationand on HDL-Associated Paraoxonase Activity CuSO₄-Induced HDL Oxidation(nmoL/mg HDL Protein) Paraoxonase Specific Activity MDA Peroxides(nmoL/mg HDL Protein/min) Control 9.1 ± 0.1 122 ± 14  26 ± 2 Atorvastatin 9.6 ± 0.5 122 ± 15  29 ± 4  Ortho-Hydroxy  0.2 ± 0.1*  9 ±1* 40 ± 4* Metabolite Para-Hydroxy  4.5 ± 0.3* 65 ± 9* 33 ± 3*Metabolite *p < 0.01 (vs. Control)

The inhibitory effects of the atorvastatin metabolites on lipoproteinoxidation is also related to a free radical scavenging activity and to ametal ion chelating capability. In the DPPH assay, a time-dependentreduction in the absorbance at 517 nm by both metabolites ofatorvastatin (20 μM), but not by atorvastatin (FIG. 5A) was observed.After 300 seconds of incubation, the ortho-hydroxy and the para-hydroxymetabolites reduced the absorbance at 517 nm by 37% and 28%,respectively. For comparison, a 95% reduction in the absorbance at wasobtained by 20 μM of the free radical scavenger antioxidant, vitamin E(FIG. 5A). These results establish that the atorvastatin metabolitespossess substantial free radical scavenging abilities.

The ability of atorvastatin metabolites to act as inhibitors of LDLoxidation by chelation of copper ions was tested by LDL incubation withincreasing concentrations of CuSO₄ to 2 hours at 37° C. in order todetermine whether excess concentrations of copper ions can overcome theinhibitory effect of these metabolites on LDL oxidation (FIG. 5B). Theaddition of increasing concentrations of copper ions to the incubationsystem caused only a minor increase in LDL oxidation when themetabolites were present, in comparison to control LDL (FIG. 5B),indicating only minimal capabilities of these metabolites to inhibit LDLoxidation via chelation of metal ions.

EXAMPLE 2

Gemfibrozil and lipoprotein oxidation

The above experiments were conducted to determine the effects ofgemfibrozil and one of its metabolites (metabolite I) on LDL oxidation,and is similar to that shown for atorvastatin (FIGS. 3-5). LDL oxidationwas inhibited by metabolite I, but not by gemfibrozil itself, in allstudied oxidative systems. This inhibitory effect of metabolite I wasconcentration-dependent (FIG. 6). At a concentration as low as 4 μM,gemfibrozil metabolite I inhibited LDL oxidation, measured by the TBARSassay, by 96% in the CuSO₄ oxidative system (FIG. 6A), by 26% in theAAPH oxidative system (FIG. 6B), and by 99% in the J-774 A.1macrophage-mediated oxidation system (FIG. 6C). Similar results werefound when LDL oxidation was analyzed by the amount of peroxides formed.The gemfibrozil metabolite I reduced LDL-associated peroxides from710±57 to 28±7 nmol/mg LDL protein in the CuSO₄ system, and from 917±78to 703±38 nmol/mg LDL protein in the AAPH system. Furthermore, the timerequired for the initiation of LDL oxidation (measured by kineticanalysis of conjugated dienes formation), revealed a lag time of 60±9minutes for LDL alone or LDL in the presence of gemfibrozil. Incontrast, even after 240 minutes of incubation with gemfibrozilmetabolite I, no conjugated diene formation in LDL was observed.

Analyses of the effect of gemfibrozil and its metabolite on VLDLoxidation again showed a very potent inhibitory effect of metabolite I(4 μM), but not of gemfibrozil, with 96% inhibition of VLDL oxidation inthe CuSO₄ oxidative system (FIG. 7A) and 91% inhibition in the AAPHoxidative system (FIG. 7B).

Lipoprotein electrophoresis of VLDL, following its oxidation withatorvastatin and its metabolites, or in the presence of gemfibrozil andits metabolite, clearly demonstrated the potency of the atorvastatinortho-hydroxy metabolite and of gemfibrozil metabolite I to reducelipoprotein electrophoretic migration (FIG. 8). Similar results wereobtained for LDL and for HDL.

Upon oxidation of HDL in the presence of 10 μM CuSO₄, metabolite I ofgemfibrozil substantially inhibited lipoprotein oxidation (Table 2),with a concomitant protection of paraoxonase activity, preserving theinitial level of HDL-associated paraoxonase activity (Table 2).Gemfibrozil itself had no effect.

Lipoprotein oxidation was carried out for 4 hours at 37° C. with 10 μMCuSO₄, in the absence (Control) or presence of 10 μM of the drugs. HDLparaoxonase activity before its incubation with the copper ions was 50±3nmol/mg HDL protein/min. Results are given as the mean±SD (n=3).

TABLE 2 The Effect of Gemfibrozil and Its Metabolites on HDL Oxidationand on HDL-Associated Paraoxonase Activity CuSO₄-Induced HDL Oxidation(nmoL/mg HDL Protein) Paraoxonase Specific Activity MDA Peroxides(nmoL/mg HDL Protein/min) Control 9.1 ± 0.1 122 ± 14  26 ± 3 Gemfibrozil 8.2 ± 0.4 134 ± 13  27 ± 5  Metabolite I  0.8 ± 0.1* 18 ± 4*50 ± 7* *p < 0.01 (vs. Control)

On analyzing the mechanisms responsible for the inhibition oflipoprotein oxidation by gemfibrozil metabolite I, both free radicalscavenging ability (FIG. 9A) and copper ion chelation capacity of thismetabolite were shown (FIG. 9B). On using the DPPH assay, onlymetabolite I, but not gemfibrozil itself (20 μM), demonstrated atime-dependent reduction in the absorbance of 517 nm, with up to 86%reduction in the optical density after 300 seconds of incubation (FIG.9A). LDL incubation with increasing concentrations of CuSO₄ for 2 hoursat 37° in the presence of gemfibrozil metabolite I revealed that onusing 20 μM CuSO₄, the inhibitory effect of metabolite I was completelyprevented (FIG. 9B), indicating that in this LDL oxidation system,chelation of copper ions by metabolite I plays a role in the inhibitionof lipoprotein oxidation.

EXAMPLE 3

Interaction Between Atorvastatin and Gemfibrozil

The general procedures described above were repeated to determinewhether the in vitro addition of the potent metabolites combined(gemfibrozil metabolite I and atorvastatin ortho-hydroxy metabolite)produces a greater inhibitory effect on LDL oxidation than either agentalone. On using low concentrations of metabolite I of gemfibrozil (3 μM)or of the ortho-hydroxy metabolite of atorvastatin (4 μM), only 40% or43% inhibitory effect of each of these drugs on copper ion-induced LDLoxidation was observed, respectively, in comparison to control LDL (FIG.10). However, on using a combination of these metabolites at the aboveconcentrations, a significant additive inhibitory effect of 88% wasobserved for LDL oxidation (FIG. 10).

EXAMPLE 4

Atorvastatin para-hydroxy metabolite, and the known antioxidants VitaminE and probucol, were evaluated in membrane vesicles enriched withpolyunsaturated fatty acids. For the lipid peroxidation experiments, 500μL of membrane vesicles were enriched with dilinoleoylphosphatidylcholine (DLPC) at a concentration of 1.0 mg DLPC/mL. Theenriched vesicles were freshly prepared in HEPES buffer(N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (0.5 mM HEPES,154.0 mM NaCl, pH 7.3). The buffer solution was prepared without addedantioxidant (as a control), and with (1) various concentrations ofatorvastatin para-hydroxy metabolite; (2) Vitamin E; and (3) probucol,which is4,4′-[(1-methyletlmylidene)bid(thio)]bis[2,6-bis(1,1-dimethylethyl)-phenol.The membrane vesicle solutions were immediately placed in a shakingwater bath at 37° C. During the incubation period (0-72 hours), 100 μLaliquot samples were removed and the peroxidation reaction wasterminated by adding 25 μL of 5.0 mM of ethylenediaminetetra acetic acid(EDTA) and 20 μL of 35.0 mM of butylated hydroxytoluene. The extent oflipid peroxidation in each sample was determined by a spectrophotometricassay for lipid peroxides in serum lipoproteins using a color reagentknown as CHOD-iodide (Merck, Darmstadt, FRG, Merck Cat. No. 14106). Thecolor reagent has the following composition:

Potassium phosphate, pH 6.2 0.2 M Potassium iodide 0.12 M Sodium azide0.15 μM Polyethyleneglyol mono[p-(1,1′,3,3′-tetramethyl-butyl-phenyl] 2g/L ether Akylbenzyldimethylammonium chloride 0.1 g/L Ammonium molybdate10 μM

The concentration of triiodide formed was measuredspectrophotometrically according to the formula (L=lipoprotein)$\begin{matrix}\left. {{LOOH} + {2H^{+}} + {2I^{-}}}\rightarrow{{LOH} + {H_{2}O} + I_{2}} \right. \\\left. {I_{2} + I^{-}}\rightarrow I_{3}^{-} \right.\end{matrix}$

To each of the withdrawn aliquots of membrane vesicles was added 1.0 mLof the CHOD color reagent, and the sample was incubated in the absenceof light for 4 hours. The absorbance of the solution was measured at 365nm (ε=2.4×10⁴ M⁻¹ cm⁻¹). Lipid peroxide formation was measured intriplicate and values were expressed as mean±SD. The significance ofdifferences between results from different experimental conditions wastested using the two-tailed student t-test.

The antioxidant activity of atorvastatin para-hydroxy metabolite isshown in FIG. 11 for various dose concentrations. The results establishthat the para-hydroxy compound has dose-dependent antioxidant activity,and at 10.0 μM causes 80% inhibition of lipid peroxidation. Even atconcentrations as low as 10.0 μM, the para-hydroxy compound inhibitedhigh levels (>10² μM) of lipid peroxidation.

The results shown in FIG. 12 establish that the atorvastatinpara-hydroxy metabolite is significantly more active than other knownantioxidants, specifically Vitamin E and probucol.

The antioxidant activity of atorvastatin para-hydroxy metaboliteincreased under atherosclerotic-like conditions of elevated membranecholesterol, and this is shown in FIG. 13.

The foregoing experiments establish that metabolites of HMG-CoAreductase inhibitors, such as atorvastatin for example, and of fibricacid derivatives, for example gemfibrozil, significantly inhibitedlipoprotein oxidation in several oxidation systems. LDL oxidation is akey event in atherogenesis, since it contributes to macrophagecholesterol accumulation and foam cell formation, as well as tocytotoxicity, thrombosis, and inflammation. Hence, inhibition of LDLoxidation contributes to attenuation of the atherosclerotic process.Although not as extensively studied, VLDL and HDL oxidation also occurunder oxidative stress, and also facilitates atherosclerosisdevelopment. In VLDL, lipid peroxidation mainly involves the oxidationof core triglyceride polyunsaturated fatty acids, whereas in HDL,surface phospholipid fatty acids are the major substrates susceptible tooxidation.

In hypercholesterolemic and in hypertriglyceridemic patients, high bloodcholesterol and triglyceride concentrations are risk factors foratherosclerosis. The increased risk is due to enhanced susceptibility ofthe lipoproteins to oxidation. Several hypolipidemic drugs have beenshown to reduce the enhanced propensity of LDL to oxidation inhypercholesterolemic patients. This inhibitory effect on LDL oxidationcould result from an enhanced removal (via drug-induced increased LDLreceptor activity, mainly in the liver) of “aged LDL” which is moreprone to oxidative modifications. In addition, this protective effectagainst oxidation may result from drug metabolites formed in vivo thatpossess antioxidant properties. However, with the exception offluvastatin, none of the parent forms of the studied hypolipidemic drugsdemonstrated a direct inhibitory effect on LDL oxidation when tested invitro at pharmacological concentrations. The above data demonstratesthat the parent drugs, atorvastatin and gemfibrozil, do not affect LDL,VLDL, or HDL oxidizability in vitro, even when used at highconcentrations. However, low pharmacological concentrations of specifichydroxylated metabolites induce very potent inhibitory effects on LDL,VLDL, and HDL oxidation, both in metal ion-dependent and -independentsystems. The drug metabolites inhibitory effect on lipoproteinoxidizability was found to be more pronounced in the CuSO₄ system, incomparison to the AAPH system, and this phenomenon may be related to theeffects of the metabolites on both scavenging of free radicals andbinding of copper ions. Both the gemfibrozil metabolite I and thehydroxy metabolites of atorvastatin were shown to be potent free radicalscavengers.

In comparison to the atorvastatin ortho-hydroxy metabolite, gemfibrozilmetabolite I acted in the CuSO₄ oxidative system as a better metal ionchelator. Increased copper ion concentrations completely abolished theinhibitory effect of gemfibrozil metabolite I, but not that of theatorvastatin metabolites, on LDL oxidation. The molecular structure ofthe atorvastatin hydroxy metabolites, where the hydroxyl group isattached to the carboxamide portion of the molecule, enable thesemetabolites to act as electron donors, and hence, as potent antioxidants(FIG. 1). The ortho-hydroxy metabolite is a more potent antioxidant thanthe para-hydroxy metabolite of atorvastatin, as the hydroxyl group inthe ortho position to the amine group (but not the hydroxyl group in thepara position), can form a relatively stable transition state of theperoxyl radical, and hence, act as a potent antioxidant. Similarly, ingemfibrozil metabolite I (but not in gemfibrozil), the hydroxyl group onthe aromatic ring can substantially contribute to the antioxidativeproperties of this compound (FIG. 2).

Under oxidative stress, lipoprotein oxidation involves the action ofreactive oxygen species, and since transition metal ions are known to bepresent in areas of the atherosclerotic lesions, the oxidation modelsused in the above experiments are representative of the in vivosituation.

The inhibitory effects of both the atorvastatin and gemfibrozilmetabolites, on LDL oxidation, were also shown for VLDL and HDL. Thepattern of inhibition was similar in all studied oxidation systems.These results establish that the metabolites exert their inhibitoryeffect on lipoprotein oxidation via common mechanisms, i.e., freeradical scavenging and metal ion chelation. In one study, in patientswith familial combined hyperlipidemia, gemfibrozil therapy did notsignificantly affect LDL oxidizability. This observation, however, couldhave resulted from too low a concentration of the drug metabolites toexert an antioxidative effect on LDL oxidation, or the time of samplecollection. In addition, drug metabolites could associate withnon-lipoprotein components of plasma (e.g. albumin) or be sequesteredwithin cells or interstitial compartments. Thus, the ex vivo examinationof oxidation potential of lipoproteins isolated from treated humans orexperimental animals may not necessarily reflect the environment of thelipoprotein in vivo.

The data presented above establishes that hydroxylated cholesterollowering agents inhibit oxidation of lipoproteins by scavenging freeradicals and by reducing metal ion chelation of lipoproteins.Accordingly, the invention provides a method for inhibiting lipoproteinoxidation, as well as a method for inhibiting metal ion chelation oflipoproteins, and a method for scavenging free radicals. The amounts ofhydroxylated cholesterol lowering agents required to inhibit metal ionchelation of lipoproteins, and to scavenge free radicals, are allreferred to herein as an “antioxidant amount”.

The hydroxylated cholesterol lowering agents will be administered in anantioxidant amount, namely an amount that is effective to cause aninhibition of lipoprotein oxidation. Such antioxidant effective amountswill be from about 1 to about 100 mg/kg. Such amounts of active agentwill be administered from one to about four times a day in order toinhibit lipoprotein oxidation.

The hydroxylated compounds will be formulated for convenient oral orparenteral administration, and will be combined with common excipientsand carriers such as calcium carbonate, candelilla wax, hydroxypropylcellulose, lactose, magnesium stearate, microcrystalline cellulose,polyethylene glycol, talc, and titanium dioxide. For oraladministration, the formulations can be pressed into tablets, orencapsulated into gelatin capsules. Typical tablets will contain fromabout 10 mg of active ingredient to about 80 mg. The compounds canadditionally be formulated as slow release dosage forms, for exampleusing osmotic pump technology, as well as transdermal skin patches. Forparenteral dosing, the compounds typically are dissolved in isotonicsaline for convenient intravenous administration, or for injection.

What is claimed is:
 1. A method for inhibiting oxidation of lipoproteinsin a mammal comprising administering an antioxidant effective amount ofa hydroxylated cholesterol lowering agent selected from a hydroxylatedstatin or a hydroxylated fibrate.
 2. A method of claim 1 employinghydroxylated gemfibrozil, hydroxylated atorvastatin, or hydroxylatedfluvastatin.
 3. A method of claim 2 employing ortho- orpara-hydroxylated atorvastatin.
 4. A method for scavenging free radicalsin a mammal comprising administering a free radical scavenging amount ofa hydroxylated cholesterol lowering agent selected from a hydroxylatedstatin or a hydroxylated fibrate.
 5. A method of claim 4 wherein thehydroxylated cholesterol lowering agent is ortho- or para-hydroxylatedatorvastatin, hydroxylated gemfibrozil, or hydroxylated fluvastatin. 6.A method for inhibiting metal ion chelation of lipoproteins in a mammalcomprising administering a metal ion chelation inhibiting amount of ahydroxylated cholesterol lowering agent selected from a hydroxylatedstatin or a hydroxylated fibrate.
 7. A method for claim 6 wherein thehydroxylated cholesterol lowering agent is ortho- or para-hydroxylatedatorvastatin, hydroxylated gemfibrozil, or hydroxylated fluvastatin.